The liver is an important target organ for gene therapy because it plays a central role in the metabolism and production of serum proteins. Nonviral vector plasmid DNA has been considered as the simplest and safest method to delivery gene to the liver. More recently, it has been demonstrated that plasmid DNA could be transfected to the liver by manually massaging the liver (MML). The method is non-invasive and does not induce any measurable toxicity in the treated animal. Furthermore, the level of gene expression is high enough to produce therapeutic effects in a diseased liver model. In this proposal, the mechanisms underlying the DNA transfer into hepatocytes by MML will be elucidated by answering specific questions: 1) Does MML transiently enlarge the fenestrae in the sinusoid endothelial cells of the liver allowing DNA to reach the hepatocytes? And 2) Does MML induce the retraction of liver endothelial cells and open the tight junctions between the liver endothelial cell, exposing the hepatocytes? To increase gene transfer efficiency, a favorable non-viral vector for MML gene transfer will be developed, which will include the synthesis of a new carrier and construction of new plasmids. The design of this vector is based on an observation that gene transfer efficiency using MML could be further enhanced if the retention time and density of plasmid DNA in the liver are increased. The carrier will be PNA-PEG-Gal, in which peptide nucleic acid (PNA) will be conjugated with polyethylene glycol (PEG), and a target ligand, galactose (Gal), appended to the distal end of PEG. The PNA-PEG-galactose carrier is designed to carry plasmid DNA to hepatocytes through receptor-mediated binding mechanism. This strategy employs galactose moieties as homing devices for hepatocytes, and a PNA as a carrier to bind sequence-specifically to DNA. As a result, the retention time and density of plasmid DNA in the liver could be increased. The plasmids, containing either a luciferase reporter or hydroxylase (PAH) genes, with an Epstein Barr virus (EBV) element/or a pBS-HCRHPI-A (liver-specific gene expression cassette, and multiple PNA-binding elements that have specific sequences for hybridization with PNA will also be constructed. The therapeutic effects on the metabolic disease on phenylketonuria (PKU) mice, will be examined after the PAH gene transfer using the approach in this proposal.